Goat anti rabbit igg

For cryo-electron microscopy, HeLa cells expressing μΔ or μΔ5 were fixed for 1 hour at room temperature (0.2% glutaraldehyde/2% paraformaldehyde in cacodylate Buffer 0.1 M) and processed as described35 (link). Briefly, samples were embedded in 12% gelatin, infiltrated in 2.3 M sucrose and frozen in liquid nitrogen. Cryosections were obtained using a Leica EM FC7 ultramicrotome (Leica microsystem, Vienna, Austria) and collected on 150 mesh formvar carbon coated copper grids. Grids were then incubated with 0.1 μg/μl rabbit anti-μ (Zymed Laboratories, San Francisco, CA) followed by goat anti-rabbit IgG coupled to 15 nm gold beads.
Grids were contrasted in a solution of uranyl acetate and methylcellulose, air-dried and observed in a Leo 912AB transmission electron microscope (Carl Zeiss, Oberkochen, Germany).
Images were analysed with ImageJ in order to determine the size of the μ-containing vesicles. At least two perpendicular measurements were performed for each structure; 60 structures were analysed for each sample and the diameter averaged.

Mouse monoclonal antibodies included; ED1 (CD68, macrophages) and R73 (rat αβ T‐cell receptor) (Serotec, Oxford, UK), RP1 (neutrophils) (Becton Dickinson, San Diego, USA) and anti‐α‐tubulin (Abcam, Cambridge, UK). Rabbit polyclonal antibodies included anti‐phospho‐p38 Thr180/Tyr182 and anti‐phospho‐c‐Jun Ser63 (Cell Signaling, Boston, MA, USA) and goat anti‐γ‐fibrinogen (Santa Cruz biotechnology, CA, USA). Biotinylated antibodies included goat anti‐mouse IgG and goat anti‐rabbit IgG (Zymed, San Francisco, CA, USA). Immunofluorescence staining used FITC (Fluorescein isothiocyanate)‐conjugated rabbit polyclonal antibodies against sheep IgG Dako, Glostrup, Denmark, rat IgG (Sigma‐Aldrich, Castle Hill, NSW, Australia) and rat C3 (Cappel, Malvern, PA, USA).